Modulation by retinoids of mRNA levels for nuclear retinoic acid receptors in murine melanoma cells

Retinoids inhibit the growth and enhance the differentiation of murine S91-C2 melanoma cells. Specific alterations in gene expression are a plausible mechanism for these effects. Since nuclear retinoic acid receptors (RAR) are likely mediators of retinoid-induced changes in gene expression, we used Northern blotting to analyze the expression of RAR alpha, RAR beta, and RAR gamma in S91-C2 cells. mRNA for both RAR alpha and RAR gamma was detected in these cells, but no RAR beta mRNA could be found. Treatment with 10(-7) and 10(-6) M beta-all-trans-retinoic acid (RA) for 24 h caused a 1.5- to 2-fold increase in RAR alpha and RAR gamma mRNA, whereas lower concentrations of RA were ineffective. RAR beta mRNA, which was undetectable in untreated cells, was detected after 24 h of treatment with a RA concentration as low as 10(-9) M, and its level increased with up to 10(-6) M RA. At the latter dose, RAR beta mRNA induction occurred by 4 h and increased progressively, reaching a plateau after 24 h of treatment. RAR beta mRNA induction at 4 h was not inhibited by cycloheximide at a concentration that suppressed protein synthesis by more than 90%. Several retinoids and related synthetic compounds, including 13-cis RA, TTNPB, Ch55, Am80, and the trifluoromethyl nonyloxyphenyl analog of RA, also induced RAR beta mRNA, whereas a 24-h treatment with 10(-6) M retinol, TTNP (a decarboxylated analog of TTNPB), or the phenyl analog of RA failed to induce RAR beta mRNA. With the exception of retinol and the trifluoromethyl nonyloxyphenyl analog of RA, the ability of the retinoids to induce RAR beta mRNA and their growth inhibitory effect were correlated. However, S91-C154, a RA-resistant mutant subclone derived from S91-C2 cells, showed mRNA levels of RAR alpha and RAR gamma and induction of RAR beta by RA similar to those detected in the sensitive S91-C2 cells. Like the S91 melanoma cells, two other mouse melanoma cell lines, K-1735P and B16-F1, constitutively expressed RAR alpha and RAR gamma mRNAs. The level of RAR beta mRNA was increased by RA only in B16-F1 cells, although the growth of both was inhibited by RA. These results demonstrate that RA can, directly and rapidly, induce the expression of mRNA for a high affinity nuclear receptor in some murine melanoma cells and that this induction is not sufficient to inhibit growth.     

Retinoic acid receptor alpha2 is a growth suppressor epigenetically silenced in MCF-7 human breast cancer cells.

Retinoic acid (RA) receptor (RAR) beta2 has been shown to be underexpressed in human breast cancer cells, including MCF-7 cells, and recent reports have suggested that hypermethylation of the RAR beta2 promoter and 5'-UTR is the underlying cause. Here we show that RAR alpha2 is also underexpressed in MCF-7 breast cancer cells, at both the message and the protein level, relative to normal or nontumorigenic breast epithelial cells. Bisulfite sequencing of the CpG island in the RAR alpha2 promoter revealed highly penetrant and uniform cytosine methylation in MCF-7 cells. Pretreatment with the DNA methyltransferase inhibitor, azacytidine, followed by treatment with RA and a histone deacetylase inhibitor, trichostatin A, resulted in partial promoter demethylation and RAR alpha2 induction, which strongly suggested that promoter hypermethylation is responsible for RAR alpha2 underexpression. We compared the outcome of ectopic expression in MCF-7 cells of matched levels of RAR alpha2 and RAR beta2. On the basis of a clonogenic assay, RAR alpha2 displayed ligand-dependent growth-suppressive activity similar to that of RARb eta2; thus, 10 and 20 nM RA inhibited clonogenic growth by 52 and 80%, respectively, in RAR alpha2-transfected cells compared with 75 and 77%, respectively, in RAR beta2-transfected cells. We conclude that the silencing of the RAR alpha2 promoter by hypermethylation may play a contributory role in the dysregulation of RA signaling in mammary tumorigenesis.     

Regulation of alpha 2(I) collagen expression in stellate cells by retinoic acid and retinoid X receptors through interactions with their cofactors

Retinoic acid (RA) suppresses alpha 2(I) collagen expression in hepatic stellate cells through the binding of retinoic acid receptor beta (RAR beta) and retinoid X receptor alpha (RXR alpha) to RA response elements (RAREs) in the alpha 2(I) collagen promoter. This study determined the influence of coactivators and corepressors to RAR beta and RXR alpha on the regulation of the alpha 2(I) collagen promoter. The coactivators, steroid receptor coactivator-1 (SRC-1) and growth hormone receptor interacting protein-1 (GRIP-1), enhanced, while the nuclear receptor corepressor (N-CoR) abolished the inhibitory effect of RAR beta and RXR alpha on the promoter activity. In the presence of RA, the coactivators SRC-1 and GRIP-1 formed complexes with RAR beta and RXR alpha which are bound to an oligonucleotide specifying a RARE site in the promoter. In conclusion, this study shows that in the presence of retinoic acid, the coactivators SRC-1 and GRIP-1 augment, while the corepressor N-CoR abolishes, the suppressive effects of RAR beta and RXR alpha on alpha 2(I) collagen promoter activity.     

Retinoic acid receptor isoform RAR gamma 1: an antagonist of the transactivation of the RAR beta RARE in epithelial cell lines and normal human keratinocytes.

The diversity of isoforms of retinoic acid (RA) receptors (RARs) and of DNA sequences of retinoic acid-responsive elements (RAREs) suggests the existence of selectivities in the RAR/RARE recognition or in the subsequent gene modulation. Such selectivities might be particularly important for RAREs involved in positive feedback, eg. the RAR beta RARE. In the present work we found that in several epithelial cell lines, reporter constructs containing the RAR beta RARE linked to the HSV-tk promoter were transactivated in the presence of RA by endogenous RARs and co-transfected RAR alpha 1 and RAR beta 2 isoforms, but not by RAR gamam 1. On the contrary, this latter isoform behaved towards the RAR beta RARE as an inhibitor of the transactivation produced by endogenous RARs and by cotransfected RAR alpha 1 and RAR beta 2. RAR gamma 1 also behaved as an antagonist of the transactivation produced by cotransfected RXR alpha. The natural RAR beta gene promoter or RAR beta RARE tk constructs were not activated by the endogenous receptors of normal human keratinocytes (NHK), which are known to contain predominantly RAR gamma 1. It was, however, possible to activate to a certain extent RAR beta RARE-reporter constructs in NHK by co-transfecting RAR alpha 1, RAR beta 2 or RXR alpha. The antagonist behavior of RAR gamma 1 towards the RAR beta RARE may explain why in certain cell types such as keratinocytes, RAR beta is neither expressed nor induced by RA.     

Overexpression of the cellular retinoic acid binding protein-I (CRABP- I) results in a reduction in differentiation-specific gene expression in F9 teratocarcinoma cells

Treatment of F9 teratocarcinoma stem cells with retinoic acid (RA) causes their irreversible differentiation into extraembryonic endoderm. To elucidate the role of the cellular retinoic acid binding protein-I (CRABP-I) in this differentiation process, we have generated several different stably transfected F9 stem cell lines expressing either elevated or reduced levels of functional CRABP-I protein. Stably transfected lines expressing elevated levels of CRABP-I exhibit an 80-90% reduction in the RA induced expression of retinoic acid receptor (RAR) beta, laminin B1, and collagen type IV (alpha 1) mRNAs at low exogenous RA concentrations, but this reduction is eliminated at higher RA concentrations. Thus, greater expression of CRABP-I reduces the potency of RA in this differentiation system. Moreover, transfection of a CRABP-I expression vector into F9 cells resulted in five- and threefold decreases in the activation of the laminin B1 RARE (retinoic acid response element) and the RAR beta RARE, respectively, as measured from RARE/CAT expression vectors in transient transfection assays. These results support the idea that CRABP-I sequesters RA within the cell and thereby prevents RA from acting to regulate differentiation specific gene expression. Our data suggest a mechanism whereby the level of CRABP-I can regulate responsiveness to RA during development.     

Variable expression of retinoie acid receptor (RARβ) mRNA in human oral and epidermal keratinocytes; relation to keratin 19 expression and keratinization potential

Previous studies have revealed that the cells that form the different regions of the oral and epidermal stratified squamous epithelia represent a number of intrinsically distinct keratinocyte subtypes, each of which is developmentally programmed to preferentially express a particular pattern of keratins and type of suprabasal histology. Retinoic acid (RA) is known to modulate stratified squamous epithelial differentiation, including expression of the basal cell keratin K19 and the suprabasal keratins K1/K10 and K4/K13. We have found that all keratinocyte subtypes are similar in their steady state levels of RAR alpha and RAR gamma mRNAs in culture and that these levels are only minimally affected by RA. In contrast, RAR beta mRNA expression varies greatly among keratinocyte subtypes and, in eight of ten cell strains examined, directly correlated with their levels of K19 mRNA. Exposure to 10(-6) M RA increases the levels of RAR beta and K19 mRNA; conversely, complete removal of RA from the medium results in reduced levels of these messages. RA does not coordinately induce RAR beta and K19 messages in nonkeratinocyte cell types: fibroblasts cultured in the presence of 10(-6) M RA express very high levels of RAR beta mRNA but do not express detectable K19, and mesothelial cells decrease their levels of RAR beta and K19 mRNA in response to 10(-6) M RA. The correlation between RAR beta and K19 mRNA levels in most keratinocyte subtypes suggests a role for RAR beta in specifying patterns of keratin expression and suprabasal differentiation in stratified squamous epithelia.     

Constitutive activation of retinoic acid receptor beta2 promoter by orphan nuclear receptor TR2

The orphan nuclear receptor TR2 functions as a constitutive activator for the endogenous retinoic acid receptor beta2 (RAR(beta2)) gene expression in P19 embryonal carcinoma cells and for reporters driven by the RAR(beta2) promoter in COS-1 cells. The activation of RAR(beta2) by TR2 is mediated by the direct repeat-5 (DR5) element located in the RAR(beta2) promoter. Furthermore, cAMP exerts an enhancing effect on the activation of RAR(beta2) by TR2, which is mediated by the cAMP response element located in the 5'-flanking region of the DR5. The constitutive activation function-1 (AF-1) of TR2 is mapped to amino acid residues 10-30 in its N-terminal A segment. A direct molecular interaction occurs between CREMtau and TR2, detected by co-immunoprecipitation, which is mediated by the N-terminal AB segment of TR2. In gel mobility shift assays, TR2 competes with P19 nuclear factor binding to the RAR(beta2) promoter, and TR2 and CREMtau bind simultaneously to this DNA fragment. The role of TR2 in the early events of RA signaling process is discussed.     

Induction of a RAR beta 2-lacZ transgene by retinoic acid reflects the neuromeric organization of the central nervous system.

The hormone retinoic acid (RA) has been implicated in the organization of the anteroposterior (AP) body axis. In this paper, we describe the effects of RA on the activity of the RA-inducible retinoic acid receptor-beta 2 (RAR beta 2) promoter. When transgenic embryos carrying a RAR beta 2-lacZ reporter gene were exposed to a single dose of RA between gestational days 8.5 to 10.5, lacZ expression was induced in the anterior central nervous system (CNS). Strikingly, the transgene was expressed in a segmented pattern reminiscent of that of Drosophila 'pair-rule' genes. RA treatment of midgastrulation embryos at day 7.5 disturbed the segmentation and produced severe craniofacial defects. We discuss the possibility that the entire anterior CNS is segmented and that this segmentation is reflected by the RAR beta 2-lacZ induction pattern.